This invention generally relates to novel compounds and methods for the detection of compounds that are agonistic or antagonistic for the binding of viral genetic material to genomic host DNA. Additionally, the inventions generally relates to compounds and methods related to gene transfer and gene therapy, as well as therapeutics for virally based diseases.
In 1872, Moritz Kaposi described a multifocal vascular tumor affecting elderly men of Mediterranean or Eastern European origin. More recently, this neoplasm has become prevalent in immunocompromised patients, such as transplant recipients on immunosuppressive therapy, and AIDS patients, where it has become the most common cancer (Beral, V. xe2x80x9cEpidemiology of Kaposi""s sarcomaxe2x80x9d Cancer Surv 10:5-22, 1991). The relationship of the disease to geography and immunocompromised patients led to the suspicion of an infectious agent in Kaposi""s sarcoma pathogenesis. This suspicion was supported when KSHV or human herpesvirus 8 (HHV8) was identified through PCR based studies of tumor samples from AIDS patients with Kaposi""s sarcoma (Chang, Y. et al. xe2x80x9cIdentification of herpesvirus-like DNA sequences in AIDS-associated Kaposi""s sarcomaxe2x80x9d Science 266:1865-1869, 1994). Subsequent studies have shown that the virus is of the gammaherpesviridae family, bearing sequence similarity to herpesvirus saimiri (HVS) and Epstein-Barr virus (EBV) (Russo, J. xe2x80x9cNucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)xe2x80x9d PNAS 93:14862-14867, 1996). Although there is increasing epidemiologic data associating the virus with human disease, little is known about the biology of this new gammaherpesvirus.
Indirect immunofluorescence studies of the latently infected BCBL cell line with serum from KS patients reveals a characteristic punctate pattern of nuclear immunofluorescence due to the presence of what was termed the latency-associated nuclear antigen (LANA) (Moore, P. S., et al. xe2x80x9cPrimary characterization of a herpesvirus agent with Kaposi""s sarcomaxe2x80x9d J Virol 70:549-558, 1996; Simpson, G. R. et al. xe2x80x9cPrevalence of Kaposi""s sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigenxe2x80x9d Lancet 348:1133-1138, 1996). LANA is detected in the majority of cells in a KS lesion as well as in cell lines derived from body cavity lymphomas (Simpson, G. R. et al. xe2x80x9cPrevalence of Kaposi""s sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigenxe2x80x9d Lancet 348:1133-1138, 1996; Rainbow, L., et al. xe2x80x9cThe 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi""s sarcoma-associated herpesvirus (human herpes 8) is encoded by orf73 and is a component of the latency-associated nuclear antigenxe2x80x9d J Virol 71:5915-5921, 1997). Studies based on the detection of antibodies to LANA have shown that KSHV infection precedes onset of KS and other associated lymphoproliferative diseases (Gao, S. J., et al. xe2x80x9cSeroconversion to antibodies against Kaposi""s sarcoma-associated herpesvirus-related latent nuclear antigens before the development of Kaposi""s sarcomaxe2x80x9d N Engl J Med 335:223-241, 1996). LANA is encoded by orf73 of KSHV and is expressed as a latency-associated protein in the infected cell (Rainbow, L., et al. xe2x80x9cThe 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi""s sarcoma-associated herpesvirus (human herpes 8) is encoded by orf73 and is a component of the latency-associated nuclear antigenxe2x80x9d J Virol 71:5915-5921, 1997; Kedes, D. H., et al. xe2x80x9cIdentification of the gene encoding the major latency-associated nuclear antigen of the Kaposi""s sarcoma-associated herpesvirusxe2x80x9d J Clin Invest 100:2606-2610, 1997). An analysis of the LANA amino acid sequence reveals several acidic and proline/glutamine rich regions as well as a zinc finger DNA binding domain (Neipel, F., et al. xe2x80x9cFleckenstein Cell-homologous genes in the Kaposi""s sarcoma-associated rhadinovirus human herpesvirus 8: determinants of its pathogenicity?xe2x80x9d J Virol 71:4187-4192, 1997; Ganem, D. xe2x80x9cKSHV and Kaposi""s sarcoma: the end of the beginning of the end?xe2x80x9d Cell 91:157-160, 1997). In spite of this suggestion that LANA may act as a transcription factor, a specific function is yet to be assigned for this viral protein.
Little is known regarding the mechanism and establishment of KSHV latency. However, the persistence of the viral genome through generations of host cell divisions potentiates the host""s propensity of contracting the disease encoded by the virus. What is needed is a drug screen for agents that would disrupt the ability of a viral genome (e.g. the KSHV genome) to bind to host DNA thereby eliminating the viral genome in the host.
The present invention generally comprises novel compounds and methods to screen for compounds that interfere with the ability of viral genomic DNA or RNA to bind to host genomic DNA. Additionally, the present invention relates to the targeting of genes to host genomes in gene therapy applications. Furthermore, the present invention relates to compositions and methods for the treatment of viral infections and tumors.
Genomic DNA from latent viruses is able to persist for multiple generations of the host cell by binding to a tethering protein that is encoded by the viral DNA. We have discovered the mechanism of binding of the viral DNA to the host cell. For example, genomic DNA from the Kaposi sarcoma virus (KSHV) is able to persist for multiple generations of the host cell by binding to a tethering protein, the latency-associated nuclear antigen (LANA). LANA tethers the KSHV viral DNA to the chromosomal structural protein, histone 1 (H1). LANA is encoded by the viral DNA, therefore it will only be present in a host cell after infection. Likewise, the lack of LANA in a host cell would indicate the lack of viral infection by viruses that utilize this or similar proteins to ensure persistence. LANA binds to specific locations on the KSHV genomic DNA designated Z6, Z8 and Z2. We have defined three other smaller binding regions that partially overlap with Z6, Z8 and Z2, which we have named LBR1 (LANA binding region 1), LBR2, LBR3 and LBR4. These regions are located at approximately 22-27 kb, 109-111 kb, 127-132 kb and a region at the left 1.8 kb of the KSHV genome including one copy of the terminal repeat, respectively. LBR1 is located within the Z1 binding region, LBR2 is located immediately 3xe2x80x2 to the Z8 binding region and LBR3 is located within the Z2 binding region. The Epstein Bar Virus (EBV) persists in host cells by a similar mechanism in that it utilizes a tethering protein (EBNA1) to bind the viral genomic DNA to host histone H1 proteins. These discoveries will permit (among other things) the screening of agents that interfere with viral DNA binding to host DNA.
As noted above, the present invention also contemplates screening assays to identify drugs that inhibit or potentiate the binding of tethering proteins (e.g. LANA and EBNA1) to host histone H1 proteins. A variety of assay formats are contemplated for testing the potential of compounds suspected of modulating tethering protein binding. In one embodiment, cells are pretreated with the compound suspected of modulating the binding of the tethering protein, followed by the addition of viral DNA or viruses that encode the tethering protein. A cell free assay for the screening of drugs that inhibit or potentiate the binding of tethering proteins (e.g. LANA and EBNA1) to host histone H1 proteins is contemplated by the present invention. For example, providing i) histone H1 proteins and ii) LANA or EBNA1; adding a compound or compounds suspected of inhibiting or potentiating the binding of LANA or EBNA1 to histone H1; and detecting said binding (e.g., by Western blot).
The invention is not limited to any particular measurement technique to measured bound tethering protein. Various methods are envisioned. In one embodiment, immunofluorescence is used. In another embodiment, immunoprecipitation is used. Compounds that inhibit tethering protein binding will reveal less bound tethering protein as compared to controls. Compounds that potentiate the binding of tethering protein will reveal increased bound tethering protein as compared to controls. The present invention also contemplates the use of high throughput screening methods. For example, the use of robotics or computer controlled systems are contemplated.
The present invention is not limited to any particular mechanism by which the viral DNA binds to the host cell. For example, the compound may inhibit the binding of the viral DNA by competitively inhibiting said binding at the binding site, by sequestering the viral DNA, or by sequestering the tethering protein.
It is not intended that the present invention be limited by the nature of the compounds to be screened in the screening assay of the present invention. For example, a variety of compounds including oligonucleotides, peptides, organic compounds and nonorganic compounds, are contemplated. Additionally, combinations of compounds are contemplated by the present invention.
It is not intended that the screening assays of the present invention be limited to any particular virus or viral genome. Many different viruses are contemplated to be used in the screening assays.
It is not intended that the screening assays of the present invention be limited to any particular host cell. Many different host cells are contemplated by the present invention to be used in the screening assays.
It is not intended that the screening assays of the present invention be limited to any particular tethering protein. Many different types of tethering proteins are contemplated to be use and detected by the present invention.
The invention contemplates compounds and methods to be used in gene directed therapy. For example, the invention contemplates the use of the LANA tethering protein (or portion thereof) in conjunction with the KSHV LANA DNA binding sites for the purpose of targeting DNA that encodes therapeutic proteins to the chromosomes of the host cell. The ability of LANA to bind histone H1, when used in combination with the a gene therapy vector containing a therapeutic protein and the specific KSHV genomic binding regions, will ensure the inclusion and persistence of the gene sequence in the host cell. It is not intended that the present invention be limited to LANA as the tethering protein. The use of other tethering proteins, such as EBNA1, is contemplated.
The invention also contemplates compounds and methods to be used in gene directed therapy where the LANA or EBNA1 protein is coupled to the DNA to be incorporated into the host is bound to the DNA by chemical means. It is not intended that the present invention be limited to a particular chemical means to bind the LANA protein to the DNA. For example, proteins may be ligated to nucleic acids via disulfide bonds (Chu, B. C. and Orgel, L. E. xe2x80x9cLigation of oligonucleotides to nucleic acids or proteins via disulfide bondsxe2x80x9d Nucl Acid Res 16:3671-3691, 1988). It is not intended that the present invention be limited to any particular sequence of DNA to which the tethering protein may be chemically linked. For example, DNA sequences without the KSHV and EBV binding sites may be used.
It is not intended that the present invention be limited to a particular gene therapy. Many different types of gene therapies are contemplated by the present invention. It is not intended that the invention be limited to any particular disease. Many diseases are envisioned as potentially treatable with the present invention. For example, multiple sclerosis, Parkinson""s disease, Huntington""s disease, diabetes and other degenerative diseases are envisioned as being candidates for treatment with the present invention.
It is not intended that the gene therapy of the present invention be limited to any particular tethering protein. Many different types of tethering proteins, such as LANA and EBNA1, are contemplated to be used by the present invention.
It is not intended that the present invention be limited by the viral DNA binding sites used in the current invention providing the tethering protein recognizes the DNA binding sites.
The present invention contemplates compounds and methods for the treatment of viral infections. For example, it is contemplated that viral vectors can be produced that encode for tethering proteins (e.g. LANA and EBNA1) mutated to bind host histone H1 with greater avidity than wild type tethering proteins but bind viral DNA binding sites with reduced avidity or do not bind viral DNA binding sites. Such viral vectors would then block access to histone H1 sites thereby preventing infectious viral DNA from being replicated along with host DNA.